We characterized a set of newborn rat brain cDNAs to the mammalian DNA replication proteins currently under study in our group. These proteins are alpha-polymerase, beta-polymerase and the single-stranded nucleic acid binding protein termed helix destabilizing protein-1. Six cDNAs for the binding protein were isolated and the nucleotide sequences of each was determined. One of these cDNAs selected for detailed study was a full-length copy of mRNA encoding the predominant species of the binding protein family in rodent tissue. This polypeptide has a molecular weight of 34,215 and is routinely purified as a truncated polypeptide of equal to 25,000-Mr. Our cDNA for rat beta-polymerase was sequenced. This DNA appeared to be a full-length mRNA copy also, in this case encoding the 40KDa enzyme. Northern blot analysis of newborn rat brain polyA+ RNA using this nick translated cDNA as probe revealed one predominant hybridizing RNA species of about the same dimension as the insert itself. Southern analysis of rat genomic DNA was consistent with a single copy gene. Similar results were obtained with human genomic DNA and the gene was localized to chromosome 8 using hybrid cell chromosome segregation techniques. Beta-polymerase levels in a number of human cell lines were determined and were found to be modestly elevated (2 to 3-fold) in lung cancer cells and in cells from patients with ataxia telangectasia. Using our cDNA for rat alpha-polymerase, Northern blot analysis of newborn rat brain polyA+ RNA revealed a single hybridizing species of mRNA of about 4600 bases. This mRNA was 4-times longer than the cDNA itself and is long enough to encode the 1800 to 19OkDa alpha-polymerase catalytic subunit described earlier. Southern blot analysis of rat genomic DNA was consistent with a single copy gene. In an attempt to obtain rat cell lines with amplified alpha-polymerase genes, we isolated cell lines resistant to the competitive inhibition of alpha-polymerase butylphenyldeoxyguanosine. Two of these cell lines were found to have increases in the amount of both alpha-polymerase enzyme activity and alpha-polymerase catalytic subunit as determined by Western blot analysis. Finally, experiments toward developing a system for study of polyoma virus DNA replication in vitro were continued.